New peptide derivatives

ABSTRACT

Tripeptide derivatives, characterized by the general formula: 
     
         R.sub.1 --X--D--Arg--A--Arg--NH--R.sub.2 
    
     wherein R 1  is hydrogen, α- or β-naphtyl residue, lower alkyl residue which may be substituted with a carboxyl group, unsubstituted or substituted phenyl- or phenylalkyl residue. ##STR1## or a single bond with the proviso that when R 1  is hydrogen then and only then X is a single bond 
     A=Gly or Sar 
     R 2  =an aromatic or heterocyclic residue which gives a compound R 2  --NH 2  by enzymatic hydrolysis, which can be determined quantitatively 
     or disalts and trisalts of inorganic or organic acids thereof, process for their preparation and method for determination of serine proteases, especially Factor X a , 
     or components which can interact with serine proteases or zymogen forms thereof.

TECHNICAL FIELD

The present invention relates to novel tripeptide derivatives useful fordetermination of serine proteases. The novel derivatives are especiallysuitable for determination of Factor X_(a) (E.C.3.4.21.6) or foranalyses of reactions, in which FX_(a) is formed, inhibited or consumedwhich makes it possible to quantify other enzymes and/or inhibitorspresent in the reactions.

THE BACKGROUND OF THE INVENTION

Factor X_(a) is a key substance in the reaction cascade which leads tothe blood coagulation. Several methods based on the determination ofFX_(a) with chromogenic substrate have shown to be of considerable valuewithin the clinical diagnostic in explanation of disturbances in theblood coagulation system (Hemker H. C.--Handbook of syntheticsubstrates, 1983, Martinus Nijhoff Publishers, Boston).

PRIOR ART

Chromogenic tetrapeptide derivatives are described by Aurell, L., et al.(Haemostasis vol. 7, 1978, 92-94) and have shown to be of considerablevalue in this case. These substrates are based on the amino acidsequence (--Ile--Glue--Gly--Arg--), which precedes the splitting placesin the natural substrate prothrombin. The tetrapeptide substrates arecharacterized by high selectivity i.e. other enzymes, especiallythrombin, do not disturb the determination of Factor X_(a).Disadvantages are however the limited solubility and the several stepsin the synthesis. EP No. 34 122 shows that tripeptide derivatives may beuseful as FX_(a) substrates. However, these tripeptides are sensitive tothrombin too. The high selectivity of the tetrapeptides is not obtainedby these tripeptides.

DESCRIPTION OF THE INVENTION

The tripeptide derivatives in the present invention show highsensitivity and solubility and simultaneously selectivity propertiescomparable with the tetrapeptides in the prior art. The novel substratesare characterized by the following formula:

    R.sub.1 --X--D--Arg--A--Arg--NH--R.sub.2

wherein R₁ is hydrogen, α- or β-naphthyl residue, lower alkyl residuewhich may be substituted with a carboxyl group, unsubstituted orsubstituted phenyl- or phenyalkyl residue, wherein the alkyl grouphaving 1 to 4 carbon atoms and preferably wherein the substitution is inpara position in the phenyl ring and the substituents are lower alkyl,lower alkoxy, halogen or nitro group;

lower alkyl is a straight or branched alkyl group having 1 to 4 carbonatoms, preferably methyl, ethyl or tert. butyl;

lower alkoxy is an alkoxy group having 1 to 4 carbon atoms, preferablymethoxy;

halogen is chlorine, bromine, fluorine, or iodine, preferably chlorine;##STR2## or a single bond with the proviso that when R₁ is a hydrogenthen and only then X is a single bond

A=Gly or Sar

R₂ =an aromatic or heterocyclic residue which gives the compound R₂--NH₂ by enzymatic hydrolysis

or di- and tri-salts of inorganic or organic acids thereof.

These compounds R₂ --NH₂ are prior known compounds with chromogenic,fluorogenic or electrochemical properties which permit quantifying ofserine proteases by determination of splitted marker directly or afterderivatization (Hemker H. C., loc. cit. and references cited therein).

Examples of compounds R₂ --NH₂ are: p-nitroaniline,3-carboxy-4-hydroxyaniline, 3-sulfo-4-nitroaniline,3-alkoxy-4-nitroaniline, 3-carboxy-4-nitroaniline,4-methoxy-β-naphtylamine, 4-(N-ethyl-N-hydroxyethyl)aminoaniline,5-amino-isophtalic acid-dimethyl ester, 5-amino-8-nitroquinoline,7-amino-4-trifluoromethyl coumarine, 7-amino-4-methyl coumarine,4-aminodiphenylamine.

The novel derivatives containing two amino acids with strong basicside-chain can form disalts or when R₁ is hydrogen trisalts withinorganic or organic acids. Preferred salts are disalts of inorganic ororganic acids since they possess a high water solubility. Especiallypreferred salts are hydrochlorides.

The splitting of the substrates by the appropriate enzymes fordetermination takes place on the carboxylic side of arginine in L-form,therefore the N-terminal arginine must be as D-isomer. Surprisingly, atbiological pH the negatively charged or neutral lipophilic part of thetetrapeptide substrate could be changed to a positively charged strongbasic amino acid with retained high selectivity and increasedsensitivity.

The use of the peptide derivatives according to the present invention inmethods for quantitative and/or qualitative determination of serineproteases or quantitative and/or qualitative determination ofcomponents, which can interact with serine proteases or zymogen formsthereof, such as Factor X which is activated to Factor Z_(a), areimportant applications.

The novel Factor X_(a) substrates constitute by their sensitivity, highsolubility and good selectivity a valuable addition at the establishmentof diagnostic analyses based on determination of Factor X_(a). A goodselectivity is an important criterion in determination of FX_(a) inplasma, since there is otherwise a risk of influence from thrombin whichmay be present initially or formed during the assay. Examples ofimportant applications are determination of FX_(a) in human plasma,after activation with a protease from Russel's Viper Venom (RVV-X), ofantifactor X_(a), of heparin, and of FVIII:C at hemophilia (Hemker H.C., loc. cit., S. Rosen, Scand. J. Haematol. Suppl. 40, vol. 33, 1984,139-145). Especially important applications are analyses in connectionwith anticoagulantia treatment with low molecular weight heparinfractions, where traditional clot-based methods cannot be used. (WalengaJ. M. et al., Seminars in Thrombosis and Hemostatis, vol. 11, no. 2,1985, 100-107). Specifically included are methods relying upondetermination of FX_(a) -activity and used for the direct determinationof FX and FX_(a) or for the indirect determination of FVII, FVII_(a),FVIII:C, FVIII:C_(a), FIX, FIX_(a), antithrombin III, platelet factor 4,heparin, low molecular weight heparins and heparinoids. The propertiesof the novel substrates make them especially suitable for use inautomatic analyzers.

DESCRIPTION OF SYNTHESIS

At the synthesis of the novel FX_(a) substrates conventional protectinggroups and coupling methods within the peptide chemistry are used (M.Bodansky: "Principles of Peptide Synthesis", Springer Verlag 1984), e.g.addition step-by-step of the amino acids at the C-terminal amino acidprovided with a marker, or synthesis of the N-terminal peptide fragmentper se, which then is coupled to the C-terminal amino acid provided witha marker.

Useful amino protecting groups are benzyloxycarbonyl-,9-fluorenylmethyloxycarbonyl- or t-butyloxy-carbonyl groups. To protectthe guanidino group of the arginine is used protonisation, anitro-protecting group or a p-toluenesulfonyl protecting group. Thecoupling between the amino acids is performed by activation of theα-carboxylic group (e.g. active esters, symmetric or asymmetricanhydrides, azide, DCCI or related reagents).

The invention will be described more in detail in the following notlimited working examples, which show the preparation of differentsubstrates according to the invention.

Purification of the intermediates and end products is performed byprecipitation, crystallization or gel filtration chromatography. Thepurified end products are lyophilized. Prefabricated glass plates ofsilica gel F₂₅₄ are used in the TLC analyses. After terminatedchromatography the plates are inspected in UV light (254 nm) and arethen developed with ninhydrin and chlorine/dicarboxidine reagent. TheR_(f) -values given are results from single chromatographies.

Solvent systems used for TLC are indicated according to the followingtable

    ______________________________________                                        Indication                                                                            Solvent system        Volume ratio                                    ______________________________________                                        A =     n-butanol: AcOH: water                                                                               (3:2:1)                                        Pa6 =   chloroform: MeOH: AcOH: water                                                                       (34:4:9:2)                                      M =     n-butanol: AcOH: water: pyridine                                                                    (15:3:12:10)                                    "3" =   EtOAc: AcOH: water: pyridine                                                                        (30:6:11:20)                                    ______________________________________                                    

HPLC analyses were performed on Merck RP column (Hibar LiChraCart) with40% MeOH in 0.5% triethylaminophosphate pH=2.35 as eluant (1 ml/min).The optical activity of the end products are determined at 589 nm in 50%acetic acid at a concentration of 0.4-1.0 g/100 ml and +25° C.

The below mentioned abbreviations have the following meaning: (IUPACindication has been used where such exists).

Amino acids:

Arg=arginine

D-Arg=D-arginine

Gly=glycine

Sar=sarcosine (N-methylglycine)

All amino acids in the substrates have L-configuration if nothing elseis indicated.

The free amino acid or peptide is indicated by H- at the N-terminalamino group and -OH at the carboxyl terminal group. The amino group isalways given to the left and the carboxyl group to the right.

ABBREVIATIONS

Ac=acetyl

AcOH=acetic acid

AMC=7-amino-4-methylcoumarine

Box=t-butyloxycarbonyl

Bs=benzenesulfonyl

Bz=benzoyl

Bzls=benzylsulfonyl

CHA=3-carboxy-4-hydroxyanilide

4-ClBs=4-chlorobenzenesulfonyl

DCCI=dicyclohexylcarbodiimide

DCU=dicyclohexylurea

DMF=dimethylformamide

Eoc=ethyloxycarbonyl

EtOAc=ethylacetate

EtOH=ethanol

Et₃ N=triethylamine

Ets=ethanesulfonyl

HOBT=1-hydroxybenzotriazole

HPLC=High Performance Liquid Chromatography

I=ionic strength

Mbs=4-methoxybenzenesulfonyl

MeOH=methanol

Mes=methanesulfonyl

Moz=4-methoxybenzyloxycarbonyl

4-Nbs=4-nitrobenzenesulfonyl

NEM=4-ethylmorpholine

4-Nz=4-nitrobenzyloxycarbonyl

Ps=β-naphtalenesulfonyl

ONp=p-nitrophenylester

pNA=p-nitroaniline

Suc=succinyl

TFA=trifluoroacetic acid

TLC=Thin Layer Chromatography

Tos=p-toluenesulfonyl

Tris=tris(hydroxymethyl)aminomethane

Z=benzyloxycarbonyl

WORKING EXAMPLES EXAMPLE 1 N.sup.α --Z--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=714.62

(1a) Boc-Gly--Arg--pNA.HBr

Molecular weight=532.40

43 mmol H--Arg--pNA.2HBr dissolved in 120 ml DMF is neutralized in cold(-10° C.) with Et₃ N. The Et₃ N.HBr formed is filtered off, whereafter43 mmol Box--Gly--OH, 45 mmol HOBT and 50 mmol DCCI are added. Thereaction goes on during stirring for 1 hour in cold and at roomtemperature overnight. The DCU formed is filtered off and the solutionis evaporated in vacuo to an oil which is dissolved in 160 ml EtOAc, iswashed with 2% NaHCO₃, H₂ O, 2% KHSO₄ and H₂ O. After drying with Na₂SO₄ the EtOAc-phase is evaporated and precipitated with diethylether.

Yield: 71%

TLC: Rf=0.23 (Pa₆).

(1b) H--Gly--Arg--pNA.TFA.HBr

Molecular weight=546.36

55 ml TFA is added to 30 mmol Boc--Gly--Arg--pNA.HBr (prepared accordingto Example 1a) dissolved in 55 ml methylenechloride. The mixture isstirred for 30 minutes at room temperature and is precipitated withdiethylether thereafter.

Yield: ˜100%

TLC: Rf=0.28 (A).

1. N.sup.α --Z--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=714.62

2.5 mmol Z--D--Arg--OH.HCl, 3 mmol HOBT and 3 mmol DCCI are added to 2.5mmol H--Gly--Arg--pNA--TFA.HBr (prepared according to Example 1b)dissolved in 25 ml DMF and neutralized in cold (-10° C.) with Et₃ N. Themixture is stirred for 1 hour in cold and for 48 hours at roomtemperature. The DCU formed is filtered off and the solution isevaporated in vacuo to an oil, 30 ml water is added and the solution iswashed with 2×20 ml EtOAc. The water phase is evaporated in vacuo, theproduct is ion exchanged on a Sephadex® QAE-25 column, in chloride formwith 50% EtOH as eluent and is purified on a Merck Lobar® prepackedcolumn (LiChroprep®.RP-8-B) with 30% MeOH as eluent (2 ml/min.) at theend. The purified product is lyophilized.

Yield: 37%

TCL: Rf=0.11 (Pa₆)

HPLC: 98.5% purity

[α]=-18.1° (c=0.5%).

EXAMPLE 2 N.sup.α --Boc--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=680.59

2.5 mmol H--Gly--ARg--pNA.TFA.Hbr and 2.5 mmol Boc--D--Arg--OH HCl aretreated with the same coupling method and reaction conditions as inexample 1.

Yield: 35%

TLC: Rf=0.55 (M)

HPLC: 96% purity

[α]=-25.5° (c=0.4%).

EXAMPLE 3 H--D--Arg--Gly--Arg--pNA.3HCl

Molecular weight=616.96

22 ml TFA is added to 12 mmol N.sup.α --Boc--D--Arg--Gly--Arg--pNA.2HCl(prepared according to example 2) dissolved in 22 ml methylenechloride.The mixture is stirred for 30 minutes at room temperature andprecipitated with diethylether thereafter. The product is ion exchangedand purified in the same way as in example 1.

Yield: 38%

TLC: Rf=0.30 (M)

HPLC: 98% purity

[α]=-62.4° (c=0.5%).

EXAMPLE 4 N.sup.α --Ets--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=672.62

0.5 mmol ethanesulfonylchloride and 80 μl Et₃ N are added to 0.5 mmolH--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)dissolved in 10 ml DMF and neutralized in cold (-10°) with 80 μl Et₃ N.The reaction goes on during stirring for 1 hour in cold and for 2 hoursat room temperature. The mixture is evaporated in vacuo. The product ision exchanged and purified in the same way as in example 1.

Yield: 44%

TLC: RF=0.5 (M)

HPLC: 9% purity

[α]=-15.6° (c=0.65%).

EXAMPLE 5 N.sup.α --Bs--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=720.66

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol benzenesulfonylchloride are treated in the same way andunder the same reaction conditions as in example 4.

Yield: 37%

TLC: Rf=0.53 (M)

HPLC: 98% purity

[α]=+11.6° (c=0.45%).

EXAMPLE 6 N.sup.α --4--Nz--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=759.64

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol 4-nitrobenzyloxycarbonylchloride are treated in the sameway and under the same reaction conditions as in example 4.

Yield: 57%

TLC: Rf=0.3 (A)

HPLC: 99% purity

[α]=-20.2° C. (c=0.45%).

EXAMPLE 7 N.sup.α --4--Nbs--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=765.66

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol 4-nitrobenzensulfonylchloride are treated in the same wayand under the same reaction conditions as in example 4.

Yield: 25%

TLC: Rf=0.28 (A)

HPLC: 97% purity

[α]=-0.8° (c=0.6%).

EXAMPLE 8 N.sup.α --Tos--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=734.69

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol p-toluenesulfonylchloride are treated in the same way andunder the same reaction conditions as in example 4.

Yield: 45%

TLC: Rf=0.25 (A)

HPLC: 98% purity

[α]=+23.1° (c=0.45%).

EXAMPLE 9 N.sup.α --Moz--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=744.66

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol 4-methyloxybenzyloxycarbonylazide are treated in the sameway and under the same reaction conditions as in example 4.

Yield: 31%

TLC: Rf=0.22 (A)

HPLC: 97% purity

[α]=-15.9° (c=0.4%).

EXAMPLE 10 N.sup.α --Mbs--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=750.69

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol 4-methoxybenzenesulfonylchloride are treated in the sameway and under the same reaction conditions as in example 4.

Yield: 45%

TLC: Rf=0.31 (A)

HPLC: 99% purity

[α]=+22.8° (C=0.4%).

EXAMPLE 11 N.sup.α --4--ClBs--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=755.12

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol 4-chlorobenzenesulfonylchloride are treated in the same wayand under the same reaction conditions as in example 4.

Yield: 31%

TLC: Rf=0.38 (A)

HPLC: 99% purity

[α]=+10.9° (c=0.4%).

EXAMPLE 12 N.sup.α --Ns--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=770.73

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol β-naphthalenesulfonylchloride are treated in the same wayand under the same reaction conditions as in example 4.

Yield: 25%

TLC: Rf=0.29 (A)

HPLC: 99% purity

[α]=-21.5° (c=0.4%).

EXAMPLE 13 N.sup.α --Z--D--Arg--Gly--Arg--AMC--.2HCl

Molecular weight=751.70

2 mmol Z--D--Arg--Gly--OH.HCl, 2.5 mmol HOBT and 2.5 mmol DCCI are addedto 2 mmol H--Arg--AMC.2HCl dissolved in 30 ml DMF and neutralized incold (-10° C.) with Et₃ N. The reaction is performed during stirring for1 hour in cold and for 24 hours at room temperature. The DCU formed isfiltered off and the solution is evaporated in vacuo to an oil, which ision exchanged and purified in the same way as in example 1.

Yield: 39%

TLC: Rf=0.7 ("3")

HPLC: 99% purity

[α]=-28.6° (c=0.5%).

EXAMPLE 14 N.sup.α --Ac--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=622.54

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol acetic acid anhydride are treated in the same way and underthe same reaction conditions as in example 4.

Yield: 43%

TLC: Rf=0.45 (M)

HPLC: 97% purity

[α]=-27.9° (c=0.4%).

EXAMPLE 15 N.sup.α --Suc--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=680.57

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol succinic acid anhydride are treated in the same way andunder the same reaction conditions as in example 4.

Yield: 40%

TLC: Rf=0.23 (A)

HPLC: 98% purity

[α]=-21.4° (c=0.5%).

EXAMPLE 16 N.sup.α --Bzls--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight 734.69

0.5 mmol H--D--Arg--Gly--Arg--pNA.3HCl (prepared according to example 3)and 0.5 mmol benzylsulfonylchloride are treated in the same way andunder the same reaction conditions as in example 4.

Yield: 38%

TLC: Rf=0.26 (A)

HPLC: 98% purity

[α]=-7.8 (c=0.4%).

EXAMPLE 17 N.sup.α --Bz--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=684.61

(17a) Z--Gly--Arg(NO₂)--pNA

Molecular weight=530.51

0.35 mol H--Arg--(NO₂)pNA.HBr dissolved in 750 ml DMF is neutralized incold (-10° C.) with Et₃ N. The Et₃ N.HBr formed is filtered off,whereafter 0.35 mol Z--Gly--OH, 0.35 mol HOBT and 0.38 mol DCCI areadded. The reaction is performed during stirring for 1 hour in cold andat room temperature overnight. The DCU formed is filtered off and thesolution is evaporated in vacuo to an oil, which is triturated with3×400 ml 2% NaHCO₃ and 500 ml water. The product is recrystallized from3.5 l MeOH.

Yield: 80%

TLC: Rf=0.6 (Pa₆).

(17a) H--Gly--Arg(NO₂)--pNA.HCl

Molecular weight=432.72

0.3 mol Z--Gly--Arg(NO₂)--pNA (prepared according to example 17a) issuspended in 630 ml AcOH. 415 ml 5.6M HBr in AcOH is added duringstirring. The mixture is stirred for 45 minutes at room temperature andpoured into 7.5 l dry diethylether during stirring. The precipitate isfiltered, washed with diethylether and dried in vacuo.

Yield: 90%

3 g product is ion exchanged in the same way as in example 1.

Yield: 73%

TLC: Rf=0.15 (Pa₆).

(17a) N.sup.α --Boc--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA

Molecular weight=697.69

4 mmol H--Gly--Arg(NO₂)--pNA.HCl (prepared according to example 17b)dissolved in 25 ml DMF is neutralized in cold (-10° C.) with Et₃ N. TheEt₃ N.HCl formed is filtered off, whereafter 4 mmolα--Box--D--Arg(NO₂)--OH, 4mmol HOBT and 4.2 mmol DCCI are added. Thereaction is performed during stirring for 1 hour in cold and at roomtemperature over the night. The DCU formed is filtered off and thesolution is evaporated in vacuo to an oil, which is suspended with 2%NaHCO₃, water, 2% KHSO₄, water and diethylether.

Yield: 73%

TLC: Rf=0.32 (Pa₆).

(17d) H--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA.HCl

Molecular weight=634.04

3 mmol N.sup.α --Box--D--Arg--(NO₂)--Gly--Arg(NO₂)--pNA (preparedaccording to example 17c) is suspended in 11 ml AcOH, 11 ml TFA is addedand the mixture is stirred for 4 hours at room temperature. The productis precipitated with diethylether and ion exchanged in the same way asin example 1.

Yield: 72%

TLC: Rf=0.61 (M)

(17e) N.sup.α --Bz--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA

Molecular weight=701.68

0.6 mmol benzoic anhydride and 70 μl Et₃ N are added to 0.5 mmolH--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA.HCl (prepared according to example17d) dissolved in 20 ml DMF and neutralized in cold (-10° C.) with 70 μlEt₃ N. The reaction is performed during stirring for 1 hour in cold andfor 2 hours at room temperature. The mixture is evaporated in vacuo andprecipitated with water. The product is suspended with 30 ml warm MeOH,is filtered and dried.

Yield: 68%

TLC: Rf=0.31 (Pa₆).

17. N.sup.α --Bz--D--Arg--Gly--Arg--pNA2HCl

Molecular weight=684.61

0.35 mmol N.sup.α --Bz--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA (preparedaccording example 17e) is deprotected by reaction with 15 ml dry HF inpresence of 0.5 ml anisole in a suitable apparatus according toSakakibara (S. Sakakibara et al. Bull. Chem. Soc., Japan vol. 240 p.7164-67, 1967) for 60 minutes at 0° C. After terminated reaction all HFis distilled and the raw product is ion exchanged and purified in thesame way as in example 1.

Yield: 36%

TLC: Rf=0.55 (M)

HPLC: 98% purity

[α]=-24.4° (c=0.6%).

EXAMPLE 18 N.sup.α --Eoc--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=652.57

(18a) N.sup.α --Eoc--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA

Molecular weight=669.63 0.6 mmol ethylchloroformate and 70 μl Et₃ N areadded to 0.5 mmol H--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA.HCl (preparedaccording to example 17d) dissolved in 20 ml DMF and neutralized in cold(31 10° C.) with 70 μl Et₃ N. The reaction goes on during stirring for 1hour in cold and for 2 hours at room temperature. The mixture isevaporated in vacuo and precipitated with water, filtrated and washedwith water and diethylether.

Yield: 89%

TLC: Rf=0.26 (Pa₆).

(18) N.sup.α --Eoc--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=652.57

0.45 mmol N.sup.α --Eoc--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA (preparedaccording to example 18a) is deprotected by reaction with 20 ml dry HFin presence of 0.5 ml anisole in the same way as in example 17.

Yield: 41%

TLC: Rf=0.55 (M)

HPLC: 98% purity

[α]=-25.2° (c=0.35%).

EXAMPLE 19 N.sup.α --Mes--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=658.60

(19a) N.sup.α --Mes--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA

Molecular weight=675.66

0.5 mmol H--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA.Hcl (prepared according toexample 17d) dissolved in 20 ml DMF and 0.6 mmol methanesulfonylchlorideare treated in the same way and under the same reaction conditions as inexample 17e.

Yield: 70%

TLC: Rf=0.47 (A).

19. N.sup.α --Mes--D--Arg--Gly--Arg--pNA.2HCl

Molecular weight=658.60

0.35 mmol N.sup.α --Mes--D--Arg(NO₂)--Gly--Arg(NO₂)--pNA (preparedaccording to example 19a) is deprotected and purified in the same way asin example 17.

Yield: 43.5%

TLC: Rf=0.48 (M)

HPLC: 97% purity

[α]=-17.6° (c=0.5%).

EXAMPLE 20 N.sup.α --Z--D--Arg--Sar--Arg--pNA.2HCl

Molecular weight=728.66

(20a) H--Sar--Arg--pNA.2HCl

Molecular weight=438.33

5 mmol Boc--SarOH, 5 mmol HOBT and 6 mmol DCCI are added to 5 mmolH--Arg--pNA.2HCl dissolved in 25 ml DMF and neutralized in cold (-10°C.) with Et₃ N. The mixture is stirred for 1 hour in cold and at roomtemperature overnight. The DCU formed is filtered off and the solutionis evaporated in vacuo to an oil, which is dissolved in 100 mln-butanol, washed with 2% NaHCO₃, H₂ O, 2% KHSO₄ and H₂ O. After dryingwith Na₂ SO₄, the n-butanol phase is evaporated and precipitated withdiethylether. The precipitate is filtrated, washed with diethylether anddried. The substance is suspended in 25 ml HCl-solution (1.5M in AcOH)and stirred for 2 hours at room temperature, precipitated withdiethylether and ion exchanged in the same way as in example 1.

Yield: 63.5%

TLC: Rf=0.15 (A).

20. N.sup.α --Z--D--Arg--Sar--Arg--pNA.2HCl

Molecular weight=728.66

3 mmol Z--D--ArgOH.HCl, 3 mmol HOBT and 3.5 mmol DCCI are added to 3mmol Sar--Arg--pNA.2HCl (prepared according to example 20a) dissolved in30 ml DMF and neutralized in cold (-10° C.) with Et₃ N. The mixture isstirred for 1 hour in cold and for 72 hours at room temperature. The DCUformed is filtered off and the solution is evaporated in vacuo. Theproduct is ion exchanged and purified in the same way as in example 1.

Yield: 43%

TLC: Rf=0.2 (A).

HPLC: 99% purity

[α]=-24.7° (c=0.55%).

EXAMPLE 21 N.sup.α --Z--D--Arg--Gly--CHA.2HCl

Molecular weight=729.65

(21a) Boc--Arg--CHA.HCl

3.2 mmol isobutyl chloroformate is added to 3.0 mmol Boc--Arg--CHA.HCldissolved in 10 ml DMF and neutralized in cold (-10° C.). The reactionmixture is stirred for 15 minutes in cold and a mixture of 3 mmol3-carboxy-4-hydroxianiline, 3 mmol NEM and 10 ml DMF are added. Thereaction goes on under stirring for 2 hours in cold and at roomtemperature overnight. The solution is evaporated in vacuo to an oil. 50ml n-butanol is added and the mixture is stirred for 3 hours at roomtemperature. The Boc--Arg--CHA.HCl formed is filtered off.

Yield: 50%

TLC: Rf=0.5 (A).

(21b) H--Arg--CHA.HCl

Molecular weight=382.25

8 ml 2N HCl in AcOH is added to 2 mmol Boc--Gly--Arg--CHA.HCl. Thereaction goes on for 45 minutes at room temperature. The reactionmixture is evaporated in vacuo to an oil, which is dissolved inisopropanol and precipitated with EtOAc.

Yield: 97%

TLC: Rf=0.15 (A).

(21c) Boc--Gly--Arg--CHA.HCl

Molecular weight=502.95

2 mmol H--Arg--CHA.2HCl dissolved in 12 ml DMF is neutralized in cold(-10° C.) with NEM, whereafter 2.1 mmol Boc--Gly--ONp is added. Thereaction goes on under stirring for 1 hour in cold and at roomtemperature overnight. The solution is evaporated in vacuo to an oilwhich is dissolved in 10 ml MeOH and precipitated with EtOAc.

Yield: 88%

TLC: Rf=0.4 (A).

(21d) H--Gly--Arg--CHA.2HCl

Molecular weight=439.30

10 ml 2N HCl in AcOH is added to 2 mmol Boc--Gly--Arg--CHA HCl. Thereaction mixture is stirred for 45 minutes at room temperature and thenprecipitated in EtOAc.

Yield 98%

TLC: Rf=0.15 (A).

(21) N.sup.α --Z--D--Arg--Gly--ARg--CHA.2HCl

Molecular weight=729.65

1 mmol H--Gly--Arg--CHA.2HCl dissolved in 10 ml DMF is neutralized incold (-10° C.) with NEM, whereafter 1.1 mmol Z--D--Arg--ONp.HNO₃ isadded. The reaction goes on for 1 hour in cold and for 2 hours at roomtemperature. The solution is evaporated in vacuo to an oil, which isprecipitated with EtOAc. The product is purified on a Sephadex® columnwith 10% AcOH as eluent and ion exchanged on a Sephadex®QAE-25 column inchloride form with 50% EtOH as eluent.

Yield: 57%

TLC: Rf=0.21 (A)

HPLC: 99% purity

[α]=-18.6° (C=0.3%).

EXPERIMENTS Determination of enzyme kinetic properties

The kinetic data for the substrates are determined by measuring theinitial velocity of hydrolysis (Vi) at different substrateconcentrations (Si) and at constant enzyme concentrations (Eo). Thebinding constant (K_(m)) and the maximum velocity (V_(max)) are obtainedin known manner from Lineweaver-Burk-plot, then the K_(cat) iscalculated (Hemker H. C. loc. cit.)

Methods

Enzymes and substrates are mixed in a buffer solution at -37° C. Theabsorbance change per minute is measured at 405 nm in aspectrophotometer (pNA-substrate) or in a spectrofluorometer atexcitation wavelength 380 nm and emission wavelength 440 nm(AMC-substrate).

The following commercially available reference substrates are used inthe tests:

Reference substrates

S-2222: Bz--Ile--Glu--(γ--OR)--Gly--ARg--pNA.HCl

R: H=50%; CH₃ =50%.

KabiVitrum AB, Stockholm, Sweden.

CBS 31.39: CH₃ SO₂ --D--Leu--Gly--Arg--pNA.AcOH

Diagnostica Stago, Asnieres s/Seine, France.

TEST 1 Determination of the Kinetic Constants for FX_(a) bovine

FX_(a) bovine (KabiVitrum) with a constant enzyme concentration

E₀ =4 nmol/l (pNA-substrate)

E₀ =2 nmol/l (AMC-substrate)

and the substrate in concentrations between 0.01-0.4 mmol/l (Si) aremixed in a trisbuffer solution 0.05 mol/l, pH=8.3, I=0.25 (NaCl) at 37°C. Absorbance change per minute is measured at 405 nm in aspectrophotometer for pNA-substrate and in a spectrofluorometer atemission wavelength 440 nm for AMC-substrate, respectively. From thesevalues then K_(m) and K_(cat) are calculated, which are shown in tableI.

Table I shows that the tripeptide substrates according to the inventionpossess a high affinity to the enzyme FX_(a) bovine simultaneously asmost of the substrates have a high rate of velocity. The selectivityconstant K_(cat) /K_(m) states that the new substrates show a betterselectivity for FX_(a) bovine than S-2222 and that even most of the newsubstrates are superior to CBS 31.39.

TEST 2 Determination of Kinetic Constants for FX_(a) Human

The substrates in concentrations between 0.1-0.7 mmol/l (Si) are mixedwith FX_(a) human plasma and treated according to the method asdescribed by Aurell; L., et al. (Perspectives in Hemostatis, Ed. FareedJ. et al, New York, Pergamon Press 1981, p. 382-388).

10 μl test plasma, control or standard and 200 μl trisbuffer solution(0.05 mol/l tris, pH 7.0, I=0.25 (NaCl)), polybrene (0.1 g/l) are mixedand incubated at 37° C. for 2-4 minutes. 200 μl substrate (37° C.) inconcentrations between 0.1-0.7 mmol/l dissolved in trisbuffer solutionprepared according to the above are added. The mixture is well stirredand within 30 seconds 200 μl RVV+CaCl₂ (20°-25° C.) are added and themixture is stirred. The absorbance change is measured at once in aspectrophotometer at 405 nm and 37° C.

The results are shown in table II.

Table II shows that the new tripeptide substrates possess a superioraffinity for enzyme FX_(a) human compared with the reference substratessimultaneously as they also show a high rate of velocity, which makesthem superior to the commercially available substrates.

TEST 3 Determination of Thrombin Sensitivity

At the determination of FX in plasma there is always a risk forformation of active thrombin in which disturbes the FX_(a)determination.

Thrombin bovine (KabiVitrum) with a constant concentration E₀ =1.3nmol/l and the substrates in concentrations between 0.1-0.4 mmol/l (Si)are mixed in a trisbuffer solution (0.05 mol/l, pH=8.3, I=0.25 (NaCl) at37° C. The test is performed according to test 1.

The absorbance change per minute is measured at 405 nm in aspectrophotometer and the K_(m) and K_(cat) -values are calculated asshown in table III.

Table III shows that the ratio for the selectivity constant FX_(a)bovine through thrombin for the new tripeptide substrates can becompared with the tetrapeptide and are superior to the known tripeptidewhen the sensitivity for FX_(a) bovine is compared to thrombin.

TEST 4 Solubility

The solubility in water and trisbuffer solution (0.05 mol/l, pH=8.3,I=0.25 (NaCl)), respectively at 25° C. for some new substrates are shownin tabel IV compared with the tetrapeptide substrate S-2222.

The table shows that the new tripeptide substrates have a clearly highersolubility in water as well as in buffer solution compared with thetetrapeptide S-2222.

                  TABLE I                                                         ______________________________________                                        Substrate K.sub.m     K.sub.cat                                                                             K.sub.cat /K.sub.m                              Ex. no.   mmol/l      sek.sup.-1                                                                            l/μmol · sek                        ______________________________________                                        S-2222    0.51        180     0.35                                             1        0.11        250     2.27                                             2        0.22        180     0.82                                             4        0.19        270     1.42                                             5        0.26        280     1.07                                             6        0.21        350     1.66                                             7        0.45        245     0.55                                             8        0.22        230     1.04                                             9        0.15        310     2.06                                            10        0.20        210     1.05                                            11        0.27        260     0.96                                            12        0.12        140     1.14                                            13        0.19        170     0.89                                            16        0.04        110     2.75                                            18        0.16        225     1.41                                            19        0.17        200     1.17                                            20        0.16        220     1.37                                            CBS 31.39 0.29        290     1.00                                            ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Substrate K.sub.m     V.sub.max V.sub.max /K.sub.m                            Ex. no.   μmol/l   μmol/l min                                                                           min.sup.-1                                    ______________________________________                                        S-2222    1970        46        0.023                                         1         380         73        0.192                                         4         340         46        0.135                                         9         210         51        0.242                                         16         50         15        0.300                                         CBS 31.39 1060        61        0.057                                         ______________________________________                                    

                  TABLE III                                                       ______________________________________                                         Ex. No.Substrate                                                                      mmol/lK.sub.m                                                                         sek.sup.-1K.sub.cat                                                                   sek1/μmol ·K.sub.cat /K.sub.m                                             ##STR3##                                     ______________________________________                                        S-2222  0.71    13      0.018   19.4                                           1      0.13    13      0.100   22.7                                           4      0.41    23      0.056   25.4                                           9      0.073   11      0.151   13.6                                          16      0.16    17      0.106   25.9                                          CBS 31.39                                                                             0.23    72      0.313    3.2                                          ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Substrate      Relative solubility                                            Ex. no.        water   tris-buffer                                            ______________________________________                                        S-2222          1       1                                                     1              >7      >5                                                     4              >7      >10                                                    9              >3      >3                                                     16             >5      >10                                                    ______________________________________                                    

We claim:
 1. Tripeptide derivative characterized by the general formula:

    R.sub.1 --X--D--Arg--A--Arg--NH--R.sub.2

wherein R₁ is hydrogen, α- or β-naphtyl residue, lower alkyl residuewhich may be substituted with a carboxyl group, unsubstituted orsubstituted phenyl- or phenylalkyl residue, wherein the alkyl grouphaving 1 to 4 carbon atoms and wherein said substituted phenyl is aphenyl substituted with a lower alkyl, lower alkoxy, halogen or nitrogroup ##STR4## or a single bond with the proviso that when R₁ ishydrogen then X is a single bond A=Gly or Sar R₂ =an aromatic orheterocyclic residue which in conjunction with the NH moiety to which itis connected gives a compound R₂ --NH₂ by enzymatic hydrolysis, whichcan be determined quantitatively,or disalts and trisalts of inorganic ororganic acids thereof.
 2. Tripeptide derivative according to claim 1characterized in that A is Gly.
 3. Tripeptide derivative according toclaim 1 characterized in that X is an oxycarbonyl- or sulfonyl group. 4.Tripeptide derivative according to claim 1 characterized in that NH₂--R₂ is a chromogenic or a fluorogenic group.
 5. Tripeptide derivativeaccording to claim 4 characterized in that NH₂ --R₂ is pNA, AMC or CHA.6. Tripeptide derivative according to claim 1 characterized in that R₁is ethyl, benzyl or tert. butyl.
 7. Tripeptide derivative according toclaim 1 in the form of disalts.
 8. Tripeptide derivative according toclaim 1 which is N.sup.α --Bzls--D--Arg--Gly--Arg--pNA.2HCl. 9.Tripeptide derivative according to claim 1 which is N.sup.α--Mos--D--Arg--Gly--Arg--pNA.2HCl.
 10. Tripeptide derivative accordingto claim 1 which is N.sup.α --Ets--D--Arg--Gly--Arg--pNA.2HCl. 11.Tripeptide derivative according to claim 1 which is N.sup.α--Z--D--Arg--Gly--Arg--pNA.2HCl.
 12. Tripeptide derivative according toclaim 1 which is N.sup.α --Boc--D--Arg--Gly--Arg--pNA.2HCl. 13.Tripeptide derivative according to claim 2 characterized in that X is anoxycarbonyl- or sulfonyl group.
 14. Tripeptide derivative according toclaim 2 characterized in that NH₂ --R₂ is a chromogenic or a fluorogenicgroup.
 15. Tripeptide derivative according to claim 3 characterized inthat NH₂ --R₂ is a chromogenic or a fluorogenic group.
 16. Tripeptidederivative according to claim 2 characterized in that NH₂ --R₂ is pNA,AMC, or CHA.
 17. Tripeptide derivative according to claim 3characterized in that NH₂ --R₂ is pNA, AMC, or CHA.
 18. Tripeptidederivative according to claim 2 characterized in that R₁ is ethyl,benzyl or tert. butyl.
 19. Tripeptide derivative according to claim 3characterized in that R₁ is ethyl benzyl or tert. butyl.
 20. Tripeptidederivative according to claim 4 characterized in that R₁ is ethyl,benzyl or tert. butyl.
 21. Tripeptide derivative according to claim 5characterized in that R₁ is ethyl, benzyl or tert. butyl.
 22. Thetripeptide derivative of claim 1 wherein said substituted phenyl issubstituted in the para position in the phenyl ring.